Wnt11 Promotes Osteoblast Maturation and Mineralization through R-spondin 2

Wnt11 signals through both canonical (β-catenin) and non-canonical pathways and is up-regulated during osteoblast differentiation and fracture healing. In these studies, we evaluated the role of Wnt11 during osteoblastogenesis. Wnt11 overexpression in MC3T3E1 pre-osteoblasts increases β-catenin accumulation and promotes bone morphogenetic protein (BMP)-induced expression of alkaline phosphatase and mineralization. Wnt11 dramatically increases expression of the osteoblast-associated genes Dmp1 (dentin matrix protein 1), Phex (phosphate-regulating endopeptidase homolog), and Bsp (bone sialoprotein). Wnt11 also increases expression of Rspo2 (R-spondin 2), a secreted factor known to enhance Wnt signaling. Overexpression of Rspo2 is sufficient for increasing Dmp1, Phex, and Bsp expression and promotes bone morphogenetic protein-induced mineralization. Knockdown of Rspo2 abrogates Wnt11-mediated osteoblast maturation. Antagonism of T-cell factor (Tcf)/β-catenin signaling with dominant negative Tcf blocks Wnt11-mediated expression of Dmp1, Phex, and Rspo2 and decreases mineralization. However, dominant negative Tcf fails to block the osteogenic effects of Rspo2 overexpression. These studies show that Wnt11 signals through β-catenin, activating Rspo2 expression, which is then required for Wnt11-mediated osteoblast maturation.Wnt signaling is a key regulator of osteoblast differentiation and maturation. In mesenchymal stem cell lines, canonical Wnt signaling by Wnt10b enhances osteoblast differentiation (1). Canonical Wnt signaling through β-catenin has also been shown to enhance the chondroinductive and osteoinductive properties of BMP2² (2, 3). During BMP2-induced osteoblast differentiation of mesenchymal stem cell lines, cross-talk between BMP and Wnt pathways converges through the interaction of Smad4 with β-catenin (2).Canonical Wnt signaling is also critical for skeletal development and homeostasis. During limb development, expression of Wnt3a in the apical ectodermal ridge of limb buds maintains cells in a highly proliferative and undifferentiated state (4, 5). Disruption of canonical Wnt signaling in Lrp5/Lrp6 compound knock-out mice results in limb- and digit-patterning defects (6). Wnt signaling is also involved in the maintenance of post-natal bone mass. Gain of function in the Wnt co-receptor Lrp5 leads to increased bone mass, whereas loss of Lrp5 function is associated with decreased bone mass and osteoporosis pseudoglioma syndrome (7, 8). Mice with increased Wnt10b expression have increased trabecular bone, whereas Wnt10b-deficient mice have reduced trabecular bone (9). Similarly, mice nullizygous for the Wnt antagonist sFrp1 have increased trabecular bone accrual throughout adulthood (10).Although canonical Wnt signaling regulates osteoblastogenesis and bone formation, the profile of endogenous Wnts that play a role in osteoblast differentiation and maturation is not well described. During development, Wnt11 is expressed in the perichondrium and in the axial skeleton and sternum (11). Wnt11 expression is increased during glucocorticoid-induced osteogenesis (12), indicating a potential role for Wnt11 in osteoblast differentiation. Interestingly, Wnt11 activates both β-catenin-dependent as well as β-catenin-independent signaling pathways (13). Targeted disruption of Wnt11 results in late embryonic/early post-natal death because of cardiac dysfunction (14). Although these mice have no reported skeletal developmental abnormalities, early lethality obfuscates a detailed examination of post-natal skeletal modeling and remodeling.In murine development, Wnt11 expression overlaps with the expression of R-spondin 2 (Rspo2) in the apical ectodermal ridge (11, 15). R-spondins are a novel family of proteins that share structural features, including two conserved cysteinerich furin-like domains and a thrombospondin type I repeat (16). The four R-spondin family members can activate canonical Wnt signaling (15, 17–19). Rspo3 interacts with Frizzled 8 and Lrp6 and enhances Wnt ligand signaling. Rspo1 enhances Wnt signaling by interacting with Lrp6 and inhibiting Dkk-mediated receptor internalization (20). Rspo1 was also shown to potentiate Wnt3a-mediated osteoblast differentiation (21). Rspo2 knock-out mice, which die at birth, have limb patterning defects associated with altered β-catenin signaling (22–24). However, the role of Rspo2 in osteoblast differentiation and maturation remains unclear.Herein we report that Wnt11 overexpression in MC3T3E1 pre-osteoblasts activates β-catenin and augments BMP-induced osteoblast maturation and mineralization. Wnt11 increases the expression of Rspo2. Overexpression of Rspo2 in MC3T3E1 is sufficient for augmenting BMP-induced osteoblast maturation and mineralization. Although antagonism of Tcf/β-catenin signaling blocks the osteogenic effects of Wnt11, Rspo2 rescues this block, and knockdown of Rspo2 shows that it is required for Wnt11-mediated osteoblast maturation and mineralization. These studies identify both Wnt11 and Rspo2 as novel mediators of osteoblast maturation and mineralization.     

Dendritic cells are stressed out in tumor

A recently paper published in Cell reports that dendritic cells (DCs) are dysfunctional in the tumor environment. Tumor impairs DC function through induction of endoplasmic reticulum stress response and subsequent disruption of lipid metabolic homeostasis.Tumors develop diverse strategies to escape tumor-specific immunity. Tumor-infiltrating dendritic cells (tDCs) are dysfunctional and/or mediate immune suppression¹. Cubillos-Ruiz et al.² showed in their recent Cell paper that tDCs exhibit an activation of the unfolded protein response (UPR), as indicated by the presence of high levels of spliced XPB1, and this may be attributed to reactive oxygen species (ROS) in tumor, which induces lipid peroxidation, leading to the endoplasmic reticulum (ER) stress in tDCs. Furthermore, they demonstrated that UPR activation in tDCs results in poor DC function, which is accompanied by impaired lipid metabolism and subsequent reduction of T cell anti-tumor immunity. Thus, these observations present a novel mechanism for tDC malfunction.ER stress is evoked by the presence of unfolded or chemically modified proteins. In short, the presence of damaged proteins is sensed by the proteins in the ER membrane. Of these, IRE1α can remove a short nucleotide sequence from mRNA encoding XBP1 protein. This splicing event facilitates XBP1 translation. XBP1 protein binds to consensus sequences in target genes and activates their expression³. Many XBP1 target genes are fatty acid synthesis enzymes. Enhanced production of fatty acids leads to the formation of lipid droplets inside the cytoplasm and extension of the ER compartment due to efficient intracellular membrane formation³. Therefore, this mechanism is a form of adaptation of the cell to the harsh environment, which sustains the production of functional proteins. In such a context, the article by Cuillos-Ruiz et al.² shows intriguing data on the XBP1 pathway in silencing DC function in the tumor environment.First, Cubillos-Ruiz et al.² observed that tDCs express high levels of spliced XBP1, its direct target genes and other markers for the ER stress response. Targeted deletion of XBP1 in CD11c⁺ DCs reveals an association of the XBP1-dependent ER stress response with immunosuppressive properties of tDCs. DC-specific deletion of XBP1 not only inhibits tumor growth and prolongs animal survival, but also reduces tumor peritoneal metastasis, ascites accumulation, and splenomegaly in an ovarian cancer-bearing mouse model.Next, the authors elucidated why the XBP1 pathway in tDCs is highly activated. Unexpectedly, neither typical tumor-associated cytokines nor hypoxia can efficiently stimulate XBP1 activation. Interestingly, tDCs contain high levels of lipid peroxidation byproducts bound to the proteins, which is associated with the production of ROS. Microarray analysis revealed that DC-specific XBP1 deletion downregulates both UPR pathway-dependent genes and lipid metabolism, which results in lower total lipid production, loss of lipid droplets in the cytoplasm and decreased production of triacylglycerides. This phenotype can be recapitulated by chemical inhibition of ROS formation or IRE1α and XBP1 signaling. Furthermore, XBP1-deficient DCs are potent stimulators of OT-1 T cells. Adoptive transfer of T cells isolated from metastatic tumor-bearing mice with DC-specific XBP1 deletion also shows their superior ability to control tumor growth.On the basis of these observations, the authors tested the effects of therapeutic intervention with the usage of nanocomplexes containing XBP1 siRNA. The size of lipid particles or nanocomplexes determines the anatomical location of specific drug delivery⁴. Additionally, they previously optimized the nanocomplexes for selective engulfing by DCs⁵. They found that administration of the nanoparticles containing XBP1 siRNA causes potent T cell activation, which is accompanied by reduced cancer metastatic foci and improved animal survival.The paper by Cubillos-Ruiz et al.² provides an important insight into DC biology in general. Since the identification of Toll-like receptor (TLR) signaling, the main direction in DC biology has been associated with PAMP and DAMP recognition in various degrees (Figure 1). However, the link between cellular metabolism in specific microenvironment and DC biology is poorly explored (Figure 1). The ER stress response has previously been observed in DCs^6,7. It is thought that in response to ER stress, XBP1 is crucial for DC generation, survival, and function^6,7. The conceptual link between XBP1 signaling and DC biology is as follows⁸: (i) stimulation of DCs leads to ER stress; (ii) ER stress activates UPR/XBP1 pathway; (iii) XBP1 activates lipid synthesis genes; (iv) lipids are used for the extension of the ER and Golgi compartment, which are of importance for cytokine production and secretion^9,10. Now, the authors challenged this concept in the context of cancer. The authors demonstrated that XBP1 signaling is strongly associated with poor T cell activation and limitation of XBP1 signaling leads to improved T cell function in the tumor environment (Figure 1). Thus, the paper sheds a new light on DC biology.Open in a separate windowFigure 1Double-faced role of XBP1 signaling pathway in DCs. Left: under immunostimulatory conditions, DCs receive signals from Toll-like receptors. NF-κB signaling induces a XBP1-dependent ER stress response, which enhances lipid metabolism. Formation of new membranes expands ER and Golgi compartments, which enhances cytokine production and secretion⁷. Right: In the tumor microenvironment, DCs are exposed to ROS, which also results in ER stress. However, in this case DC lipid metabolism is impaired and DCs acquire an immunosuppressive phenotype².Of course, as with any interesting work, the article raises more questions than answers. For example, which immune-related properties of DCs are “selectively” affected by the XBP1 pathway? Is there an actual cause-and-effect relationship between aberrant lipid accumulation and the immunosuppressive phenotype of tDCs? The authors observed no obvious change in PD-L1 (B7-H1) expression in DCs, but noticed reduced surface levels of peptide-loaded MHC-I complexes in wild-type tDCs as compared to XBP-deficient tDCs. However, it remains unknown whether and how DC cross-presentation is involved in tDC function regulated by ER stress, XBP1 activation, and lipid metabolism. Human ovarian cancer-associated DCs express high levels of B7-H1 and limited IL-12¹¹. It would be interesting to thoroughly examine the cytokine profile, and the B7 and TNF family members, along with lipid pathway manipulation in XBP1^−/− DCs. It is well known that ER morphology and function is substantial for the synthesis of membrane proteins and cytokines, which would be affected by the XBP1 pathway. Paradoxically, a restricted ER stress response can help immune reaction, which requires the expansion of the ER compartment. Another question is how and which lipid synthesis and metabolism pathway is targeted by XBP1 in DCs (or/and tumor cells in the same environment). Obviously, future studies are warranted to address these important questions.In conclusion, the paper by Cubillos-Ruiz et al.² opens an interesting chapter for scientifically and therapeutically exploring DC biology in a specific metabolic environment. Given that silencing XBP1 signaling in DCs enhances tumor immunity, and XBP1 is an intrinsic pro-tumor factor, it is reasonable to assume that targeting this pathway may be beneficial in patients with cancer and could kill two birds with one stone.     

Enlightening the impact of immunogenic cell death in photodynamic cancer therapy

EMBO J 31 5, 1062–1079 (2012); published online January172012In this issue of The EMBO Journal, Garg et al (2012) delineate a signalling pathway that leads to calreticulin (CRT) exposure and ATP release by cancer cells that succumb to photodynamic therapy (PTD), thereby providing fresh insights into the molecular regulation of immunogenic cell death (ICD).The textbook notion that apoptosis would always take place unrecognized by the immune system has recently been invalidated (Zitvogel et al, 2010; Galluzzi et al, 2012). Thus, in specific circumstances (in particular in response to anthracyclines, oxaliplatin, and γ irradiation), cancer cells can enter a lethal stress pathway linked to the emission of a spatiotemporally defined combination of signals that is decoded by the immune system to activate tumour-specific immune responses (Zitvogel et al, 2010). These signals include the pre-apoptotic exposure of intracellular proteins such as the endoplasmic reticulum (ER) chaperon CRT and the heat-shock protein HSP90 at the cell surface, the pre-apoptotic secretion of ATP, and the post-apoptotic release of the nuclear protein HMGB1 (Zitvogel et al, 2010). Together, these processes (and perhaps others) constitute the molecular determinants of ICD.In this issue of The EMBO Journal, Garg et al (2012) add hypericin-based PTD (Hyp-PTD) to the list of bona fide ICD inducers and convincingly link Hyp-PTD-elicited ICD to the functional activation of the immune system. Moreover, Garg et al (2012) demonstrate that Hyp-PDT stimulates ICD via signalling pathways that overlap with—but are not identical to—those elicited by anthracyclines, which constitute the first ICD inducers to be characterized (Casares et al, 2005; Zappasodi et al, 2010; Fucikova et al, 2011).Intrigued by the fact that the ER stress response is required for anthracycline-induced ICD (Panaretakis et al, 2009), Garg et al (2012) decided to investigate the immunogenicity of Hyp-PDT (which selectively targets the ER). Hyp-PDT potently stimulated CRT exposure and ATP release in human bladder carcinoma T24 cells. As a result, T24 cells exposed to Hyp-PDT (but not untreated cells) were engulfed by Mf4/4 macrophages and human dendritic cells (DCs), the most important antigen-presenting cells in antitumour immunity. Similarly, murine colon carcinoma CT26 cells succumbing to Hyp-PDT (but not cells dying in response to the unspecific ER stressor tunicamycin) were preferentially phagocytosed by murine JAWSII DCs, and efficiently immunized syngenic BALB/c mice against a subsequent challenge with living cells of the same type. Of note, contrarily to T24 cells treated with lipopolysaccharide (LPS) or dying from accidental necrosis, T24 cells exposed to Hyp-PDT activated DCs while eliciting a peculiar functional profile, featuring high levels of NO production and absent secretion of immunosuppressive interleukin-10 (IL-10) (Garg et al, 2012). Moreover upon co-culture with Hyp-PDT-treated T24 cells, human DCs were found to secrete high levels of IL-1β, a cytokine that is required for the adequate polarization of interferon γ (IFNγ)-producing antineoplastic CD8⁺ T cells (Aymeric et al, 2010). Taken together, these data demonstrate that Hyp-PDT induces bona fide ICD, eliciting an antitumour immune response.By combining pharmacological and genetic approaches, Garg et al (2012) then investigated the molecular cascades that are required for Hyp-PDT-induced CRT exposure and ATP release. They found that CRT exposure triggered by Hyp-PDT requires reactive oxygen species (as demonstrated with the ¹O₂ quencher L-histidine), class I phosphoinositide-3-kinase (PI3K) activity (as shown with the chemical inhibitor wortmannin and the RNAi-mediated depletion of the catalytic PI3K subunit p110), the actin cytoskeleton (as proven with the actin inhibitor latrunculin B), the ER-to-Golgi anterograde transport (as shown using brefeldin A), the ER stress-associated kinase PERK, the pro-apoptotic molecules BAX and BAK as well as the CRT cell surface receptor CD91 (as demonstrated by their knockout or RNAi-mediated depletion). However, there were differences in the signalling pathways leading to CRT exposure in response to anthracyclines (Panaretakis et al, 2009) and Hyp-PDT (Garg et al, 2012). In contrast to the former, the latter was not accompanied by the exposure of the ER chaperon ERp57, and did not require eIF2α phosphorylation (as shown with non-phosphorylatable eIF2α mutants), caspase-8 activity (as shown with the pan-caspase blocker Z-VAD.fmk, upon overexpression of the viral caspase inhibitor CrmA and following the RNAi-mediated depletion of caspase-8), and increased cytosolic Ca²⁺ concentrations (as proven with cytosolic Ca²⁺ chelators and overexpression of the ER Ca²⁺ pump SERCA). Moreover, Hyp-PDT induced the translocation of CRT at the cell surface irrespective of retrograde transport (as demonstrated with the microtubular poison nocodazole) and lipid rafts (as demonstrated with the cholesterol-depleting agent methyl-β-cyclodextrine). Of note, ATP secretion in response to Hyp-PDT depended on the ER-to-Golgi anterograde transport, PI3K and PERK activity (presumably due to their role in the regulation of secretory pathways), but did not require BAX and BAK (Garg et al, 2012). Since PERK can stimulate autophagy in the context of ER stress (Kroemer et al, 2010), it is tempting to speculate that autophagy is involved in Hyp-PDT-elicited ATP secretion, as this appears to be to the case during anthracycline-induced ICD (Michaud et al, 2011).Altogether, the intriguing report by Garg et al (2012) demonstrates that the stress signalling pathways leading to ICD depend—at least in part—on the initiating stimulus (Figure 1). Speculatively, this points to the coexistence of a ‘core'' ICD signalling pathway (which would be common to several, if not all, ICD inducers) with ‘private'' molecular cascades (which would be activated in a stimulus-dependent fashion). Irrespective of these details, the work by Garg et al (2012) further underscores the importance of anticancer immune responses elicited by established and experimental therapies.Open in a separate windowFigure 1Molecular mechanisms of immunogenic cell death (ICD). At least three processes underlie the immunogenicity of cell death: the pre-apoptotic exposure of calreticulin (CRT) at the cell surface, the secretion of ATP, and the post-apoptotic release of HMGB1. ICD can be triggered by multiple stimuli, including photodynamic therapy, anthracycline-based chemotherapy, and some types of radiotherapy. The signalling pathways elicited by distinct ICD inducers overlap, but are not identical. In red are indicated molecules and processes that—according to current knowledge—may be required for CRT exposure and ATP secretion in response to most, if not all, ICD inducers. The molecular determinants of the immunogenic release of HMGB1 remain poorly understood. ER, endoplasmic reticulum; P-eIF2α, phosphorylated eIF2α; PI3K, class I phosphoinositide-3-kinase; ROS, reactive oxygen species.